Abstract: The ability for pathogens to rapidly develop resistance to our best antimicrobials makes for a formidable treatment challenge, with devastating consequences to human health. The problem has been further exacerbated by the innovation gap in the development of new antimicrobials, partially resulting from the belief that resistance to new drugs is inevitable. The combination of a rising tide of resistance and the lack of novel approaches to the problem makes for a particularly dire situation. In response to this need, we seek to fundamentally alter the paradigm for combating drug resistance by targeting the very pathways that allow pathogens to mutate and evolve drug resistance. This impact of multidrug resistant pathogens is well exemplified by Pseudomonas aeruginosa, an organism that can become increasingly drug resistant through the disease course of cystic fibrosis, often greatly limiting therapeutic options and contributing to mortality. Evolvability and drug resistance in P. aeruginosa are tied to the pathway that governs stress responses, known as the SOS pathway. In our proposed research program, we aim to target key regulatory and effector enzymes, LexA and DinB, involved in this pro-mutagenic pathway. In its basal state, the regulatory protein LexA, a bi-functional repressor-protease, maintains stress response in an off state. When stress is sensed, LexA cleaves itself to turn on stress responses. As part of this response, DinB and other error-prone DNA polymerases are induced promoting the introduction of increased levels of mutations in the genome. Our strategy is to interrogate and perturb both ends of the SOS response. We propose to use chemical biology, enzymology and biophysical techniques to explore the substrate preferences for these enzymes and translate these findings into the discovery of small molecules that can perturb these enzymes of evolution. After establishing the molecular basis for LexA auto-proteolysis, we will uncover inhibitors of self-cleavage that can prevent the SOS switch from being flipped on. In parallel, we propose to define the open active site of the error-prone polymerase DinB. We will exploit its tolerance to discover nucleotide analogs which can specifically inhibit this evolutionary polymerase. With chemical probes at hand, we will directly evaluate the impact of anti-evolutionary small molecules on the acquisition of drug resistance in P. aeruginosa. Our proposed studies will help advance our understanding of how mutations arise in bacterial pathogens, a question of fundamental scientific importance. In this manner, our investigations will shape how we think about the balanced requirements for stability and adaptability in the genome. Most importantly, our studies address the exigent need for innovative alternative approaches to combating the scourge of drug resistance. Public Health Relevance: The mounting public health problem of drug-resistant pathogens is yet more menacing given the innovation gap in the discovery of new antimicrobial drugs. We propose to pursue an innovative approach to this problem, by targeting the very pathways that allow a pathogen to adapt, evolve and thereby acquire drug resistance.